A new paradigm of gene expression regulation has emerged recently with the discovery of microRNAs (miRNAs), an evolutionary conserved class of small (approximately 22 nucleotide -nt-), regulatory RNAs. miRNAs bind with partial or extensive complementarity to their mRNA targets, miRNAs control gene expression by repressing the translation or by destabilizing, by endonucleolytic cleavage, their mRNA targets, miRNAs may exert profound effects in gene expression regulation as they have the capacity to target numerous mRNAs, miRNAs are functionally equivalent to small interfering RNAs (siRNAs). The use of synthetic siRNAs to knockdown gene expression has already transformed basic biology research and is promising to revolutionize the practice of medicine, if issues regarding delivery and specificity of siRNAs are solved. Despite the recent, explosive growth in the mi/siRNA field, many important questions regarding the function of mammalian miRNAs remain unanswered and form the basis of this proposal. In Aim 1, we will identify and characterize the mi/siRNA-guided endoribonuclease (RNAi endoribonuclease). We present the partial affinity purification and properties of this enzyme and biochemical approaches that will allow us to identify and characterize this important enzyme. We also present results and strategies to elucidate how mi/siRNAs recognize their mRNA targets. In Aim 2, we will analyze the factors and mechanisms underlying miRNA-directed translational repression. We have begun the identification of proteins that miRNAs associate with, when they recognize their cognate mRNA targets in polyribosomes, and we have generated an in vitro system that recapitulates mammalian miRNA-directed translational repression. We present approaches to characterize translational repression mediated by mammalian miRNAs. We expect that our studies will promote our understanding of the molecular mechanisms underlying RNAi and the function of miRNAs in humans.